Identification of lipid-accessible sites on the Nephrops 16-kDa proteolipid incorporated into a hybrid vacuolar H+-ATPase: Site-directed labeling with N-(1-pyrenyl)cyclohexylcarbodiimide and fluorescence quenching analysis

Proton translocation by the vacuolar Hf-ATPase is mediated by a multicopy t ransmembrane protein, the 16-kDa proteolipid. It is proposed to assemble in the membrane as a hexameric complex, with each polypeptide comprising four transmembrane helices. The fourth helix of the proteolipid contains an int ramembrane acidic residue (GIu140) which is essential for proton translocat ion and is reactive toward N,N'-dicyclohexylcarbodiimide (DCCD). Current th eoretical models of proton translocation by the vacuolar ATPase require tha t Glu140 should be protonated and in contact with the membrane lipid. In th is study we present direct support for this hypothesis. Modification with t he fluorescent DCCD analogue N-(1-pyrenyl)cyclohexylcarbodiimide, coupled t o fluorescence quenching studies and bilayer depth measurements using the p arallax method, was used to probe the position of Glu140 with respect to th e bilayer. Glutamate residues were also introduced mutagenically as targets for the fluorescent probe in order to map additional lipid-accessible site s on the 16-kDa proteolipid. These data are consistent with a structural mo del of the 16-kDa proteolipid oligomer in which the key functional residue Glu140 and discrete faces of the second and third transmembrane helices of the 16-kDa proteolipid are exposed at the lipid-protein interface.